WO2009150865A1 - Method for production of modified protein and modified protein produced by the method, and protein modification kit - Google Patents

Abstract

Disclosed is a method for producing a modified protein. In the method, a Pictet-Spengler reaction is carried out between a protein containing an ethylamine group having an aromatic ring or a heteroaromatic ring at β-position and a modification molecule having an aldehyde group at the terminal of a modification unit therein, between a protein having an aldehyde group and a modification molecule containing, at the terminal of a modification unit therein, an ethylamine group having an aromatic ring or a heteroaromatic ring at β-position, or between a protein having an aldehyde group and a modification molecule comprising ethylamine having an aromatic ring or a heteroaromatic ring at β-position under weakly acidic to weakly basic conditions, thereby modifying the protein with the modification molecule which has been ring-opened.

Description

Method for producing modified protein, modified protein obtained by this production method, and protein modification kit

The present invention relates to a method for producing a modified protein. More specifically, the present invention relates to a method for producing a modified protein capable of modifying a protein in a position-specific manner while suppressing the risk of protein degradation and denaturation, and the production method. And a kit for obtaining the modified protein.
This application claims priority based on Japanese Patent Application No. 2008-155994 filed in Japan on June 13, 2008, the contents of which are incorporated herein by reference.

Currently, in designing and producing a protein having an unprecedented useful property, a modified protein in which a desired modified molecule is artificially introduced into the protein is attracting attention. Examples of a method for introducing the desired modified molecule into a protein include introduction of an unnatural amino acid using a protein translation system and de novo chemical synthesis. However, each of these methods has the following limitations.

Non-natural amino acids can be introduced in protein biosynthesis by a mutant aminoacyl tRNA synthetase; a suppressor tRNA capable of binding to the non-natural amino acid in the presence of the mutant aminoacyl tRNA synthetase; and nonsense at a desired position. There is known a method of expressing a protein containing an unnatural amino acid using a mutated gene; and the like (see, for example, Non-Patent Documents 1 to 11). This method is an excellent method in that the specificity of the introduction position of the unnatural amino acid is high.
However, this method depends on the modification of the substrate specificity of biomolecules involved in protein synthesis or the diversity of chemical structures of amino acids. Therefore, the types of unnatural amino acids that can be introduced are restricted by themselves. That is, this method cannot introduce non-natural amino acids having a structure exceeding the limit of diversity. In addition, even if an unnatural amino acid with significantly different properties and sizes compared to the original amino acid can be introduced, the presence of this unnatural amino acid prevents normal folding of the protein at the translation stage. The possibility of obstructing cannot be denied.

Another example is de novo chemical synthesis, but in this method, it is difficult to synthesize a protein having 100 or more amino acids, and there is no guarantee that the protein will be folded correctly.

As another example, a method of modifying a protein by chemical modification is also known. However, there is a problem that the position at which the chemical functional group is introduced cannot be specified depending on the chemical reaction used when modifying the protein. For example, when an amino group in a protein is modified by a chemical modification method, the N-terminus and all lysine residues are modified.

Furthermore, a method of performing chemical modification after introducing an unnatural amino acid into a protein (post-translational modification method) has also been attempted. Non-Patent Document 12 describes a method of reacting using a protein containing azidohomoalanine, a phosphine reagent, and Staudinger ligation. However, nitrene produced by the decomposition of azide reacts with the amino group of protein lysine. Similarly, a method of introducing a keto group on the protein side and reacting this keto group with a functional molecule containing an amine or hydrazide is also known (see Non-Patent Documents 13 and 14). However, although hydrazone formation and oximation are somewhat stable, hydrolysis proceeds little by little. In addition, a method has been reported in which a diene group is introduced on the protein side and an olefin reagent is introduced by a Diels-Alder reaction (see Non-Patent Document 15). However, in this method, it is difficult to introduce site-specific dienes into proteins, and side reactions with cysteine thiols proceed.

In addition, a method of chemically modifying a protein by a chemical reaction using a transition metal catalyst has been reported (Non-Patent Documents 16 to 18). However, this method requires complicated experimental operations and requires detailed examination of conditions. Furthermore, there is a risk of protein denaturation due to metal ions.

On the other hand, as a carbon-carbon bond forming reaction with high specificity, the Pictet-Spengler reaction is widely known (see Non-Patent Documents 19 to 21). The Pictet-Spengler reaction is a reaction in which ethylamine having an aromatic ring or heteroaromatic ring at the β-position and an aldehyde are subjected to ring-closing condensation, and is widely used for the synthesis of heterocyclic compounds. This reaction is usually carried out under organic solvent conditions (see Non-Patent Document 19), and the reaction proceeds even in an aqueous solvent. In that case, 10% TFA aqueous solution is used as a catalyst (refer nonpatent literature 20). However, protein denaturation cannot be prevented under these conditions. It is also described that indole-3-ethylamine having a nucleophilic property such as tryptamine proceeds even under physiological conditions (see Non-Patent Document 21). In this case, it is necessary to use an aldehyde with low steric hindrance for the reaction, and no protein or protein application is described.

A ligation reaction of oligopeptides using this Pictet-Spengler reaction has been reported (see Non-Patent Document 22). This method does not prevent protein denaturation because ligation is performed using a strong acid of 1% aqueous solution of TFA. In addition, since monoaldehyde having low reactivity is used, the modification reaction does not proceed in a 50% acetic acid aqueous solution, and the protein modification reaction cannot be performed under mild conditions.

On the other hand, methods for site-specific aldehyde formation of proteins have been reported (see Non-Patent Documents 23 and 24). In the former method (Non-Patent Document 23), a protein containing a specific primary sequence is expressed, and an aldehyde is prepared by enzymatic oxidation of the side chain of the cysteine residue. However, a highly reactive activated aldehyde cannot be prepared, and only hydrazone formation and oxime formation which are unstable in an aqueous solvent are performed. On the other hand, the latter (Non-patent Document 24) can prepare activated α-ketoaldehyde by oxidative deamination reaction using pyridoxal-5-phosphate. However, only unstable oximation is carried out in an aqueous solvent, and no application to the Picte-Spengler reaction type modification reaction is described.
Koide et al., Proceedings of the National Academy of Sciences, USA, 85, 1988, p. 6237-6241 Noren et al., Science, 244, 1989, p. 182-8 Wang et al., Science, 292, 2001, p. 498-500 Chin et al., Journal of American Chemical Society, vol. 124, 2002, p. 9026-9027 Chin et al., Proceedings of the National Academy of Sciences, USA, Vol. 99, 2002, p. 11020-11024 Furter, Protein Sci., Wang et al., Journal of American Chemical Society, vol. 124, 2002, p. 1836-1837 Santoro et al., Nature Biotechnology, Kiga et al., Proceedings of the National Academy of Sciences, USA, Vol. 99, 2002, p. 9715-9723 Sakamoto et al., Nucleic Acids Res., Volume 30, 2002, p. 4692-4699 Kirschenbaum et al., ChemBioChem 2002, No. 02-03, 235-237 Kiick et al., Proceedings of the National Academy of Sciences, USA, Vol. 99, 2002, p. 19-24 Datta et al., Journal of American Chemical Society, vol. 124, 2002, p. 5652-5653 Liu et al., Journal of American Chemical Society, Vol. 125, 2003, p. 1702-1703 Araujo et al., Angewandte Chemie International Edition, Volume 45, 2006, p. 296-301 Kodama et al., ChemBioChem, Kodama et al., ChemBioChem, Vol. 8, 2007, p. 232-238 McFarland et al., Journal of American Chemical Society, Vol. 127, 2005, p. 13490-13491 Eric et al., Chemical Review (Chem. Rev.), 95, 1995, p. 1797-1842 Biswajit et al., Tetrahedron Letters, 48, 2007, p. 1379-1383 Whaley et al., Organic Reaction, Vol. 6, 1951, p. 151-190 Li et al., Tetrahedron Letters, 41, 2000, p. 4069-4073 Carrico et al., Nature Chemical Biology, Gilmore et al., Angewante Chemie International Edition (Angew. Chem. Int. Ed.), 45, 2006 p. 5307-5311

The present invention has been made in view of the above circumstances, and introduces a desired modified molecule in a position-specific manner within a protein under milder conditions rather than reaction conditions that cause degradation or denaturation of a protein by a strong acid. An object of the present invention is to provide a method for producing a modified protein.

The present invention employs the following means in order to solve the above problems and achieve the object.
(1) The method for producing a modified protein of the present invention comprises a protein containing an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position, and a modified molecule having an aldehyde group at the end of the modifying unit. Containing protein and a modified molecule having an ethylamine group having an aromatic ring or heteroaromatic ring at the β-position at the end of the modifying unit, or a protein containing an aldehyde group and an aromatic ring or heteroaromatic ring at the β-position A modified molecule composed of ethylamine having a Pichite-Spengler reaction under a weakly acidic to weakly basic condition to modify the protein with the modified molecule closed.
(2) The aromatic ring or heteroaromatic ring preferably has an electron donating functional group.
(3) It is preferable that the electron-donating functional group is one or more selected from the group consisting of an alkyl group, an alkoxy group, an amino group, and a hydroxy group.
(4) It is preferable that the carbon atom adjacent to the amine group of the ethylamine group has an electron-withdrawing functional group.
(5) It is preferable that the carbon atom adjacent to the aldehyde group has an electron-withdrawing functional group.
(6) It is preferable that the electron-withdrawing functional group is one or more selected from the group consisting of an imino group, a carbonyl group and a carboxyl group.
(7) The ethylamine group having an aromatic ring or a heteroaromatic ring preferably has a tryptamine skeleton or a dopamine skeleton, and the aldehyde group preferably has an α-ketoaldehyde.
(8) Preferably, the protein containing an aldehyde group can be introduced with an aldehyde group by oxidative deamination reaction in the presence of pyridoxal-5-phosphate.
(9) The protein has a nonsense mutation so that it has a suppressor tRNA bound to an amino acid containing an ethylamine group or an aldehyde group having an aromatic ring or a heteroaromatic ring at the β-position, and a codon corresponding to the codon of the suppressor tRNA. It is preferably a protein synthesized by a synthesis system using the applied gene.
(10) The protein containing an ethylamine group having an aromatic ring or heteroaromatic ring at the β-position is preferably a protein having tryptophan at the N-terminus.

(11) The modified protein of the present invention can be obtained by the method for producing a modified protein described in (1) above.
(12) The protein is preferably represented by any one of the following general formulas (1) to (6).

In the general formula (1), R ¹ is a protein, R ⁵ is a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and at least one of R ² , R ³ , and R ⁴ One is the modification unit, the rest are R ² is an imino group, a carbonyl group, and a carboxyl group, and R ³ and R ⁴ are each independently a hydrogen atom, an alkyl group, a hydroxy group, an alkoxy group, or an amino group. is there.

In the general formula (2), R ¹ is a protein, R ⁵ is a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and at least one of R ² , R ³ , and R ⁴ One is the modification unit, the rest are R ² is an imino group, a carbonyl group, and a carboxyl group, and R ³ and R ⁴ are each independently a hydrogen atom, an alkyl group, a hydroxy group, an alkoxy group, or an amino group. is there.

In the general formula (3), R ¹ is a protein, R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁵ and R ⁷ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁵ , and R ⁷ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group.

In the general formula (4), R ¹ is a protein, R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁵ and R ⁷ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁵ , and R ⁷ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group.

In the general formula (5), R ¹ is a protein, R ⁵ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁴ and R ⁶ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁴ , and R ⁶ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group.

In the general formula (6), R ¹ is a protein, R ⁵ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁴ and R ⁶ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁴ , and R ⁶ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group.

The protein modification kit of the present invention is a protein modification kit for producing a modified protein, comprising pyridoxal-5-phosphate and a modified molecule containing an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position. Have.

According to the method for producing a modified protein described in (1) above, site-specific control in post-translational modification of a protein becomes possible. Since this method can introduce various modified molecules into proteins, the modified proteins can have diversity. In addition, this method produces little by-products, can be modified even at low concentrations of the substrate, and boasts high modification efficiency. In addition, since no catalyst is required, in vivo experiments are possible. Furthermore, the protein modification operation is simple, the modification is possible with an inexpensive reagent, and no large-scale equipment is required. Therefore, it is possible to construct a mass production system or a high-throughput system for the modified protein.
Extending the functional properties of proteins through new modification methods will contribute to many areas in the future, including functional proteomics and nanotechnology. The method also enables the production of designed modified proteins.

FIG. 1 is a diagram schematically showing an example of a method for producing a modified protein of the present invention. FIG. 2 is a diagram schematically showing an example of a modified protein into which a fluorescent molecule has been introduced. FIG. 3 is a diagram schematically showing an example of a modified protein into which a fluorescent group has been introduced. FIG. 4 is a diagram schematically showing an example of a modified protein into which liquid crystal molecules are introduced. FIG. 5 is a diagram schematically showing an example of a modified protein having a heavy atom introduced therein. FIG. 6 is a diagram schematically showing the production process of the modifying molecule used in Example 1. FIG. 7 is a diagram schematically showing the production process of the modified protein in Example 1. FIG. 8 is a diagram showing the results of cyprotangerin staining and Western blotting in the modified proteins of Example 1 and Comparative Example. FIG. 9 is a diagram showing measurement results using mass spectrometry for the modified protein of Example 2. FIG. 10A is a diagram showing the modified protein produced in Example 2. FIG. 10B is a diagram showing the modified protein produced in Example 2. FIG. 11 is a diagram showing the results of MS / MS analysis in the modified protein of Example 2. FIG. 12 shows the results of UV measurement for the modified protein of Example 2. FIG. 13 is a diagram showing the results of CD measurement for the modified protein of Example 2.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Formylated myoglobin 2 Tryptamine 3 Tryptamine modified myoglobin 4 β-carboline modified myoglobin 5 Modified molecule containing fluorescein 6 Fluorescein modified myoglobin 7 Modified molecule containing Alexafluoro488 8 Modified molecule 10 containing Alexafluoro488 modified myoglobin 9 MBBA MBBA modified myoglobin 11 modified molecule 12 containing bromine atom heavy element modified myoglobin 13 biotin 14 tryptophan 15 modified molecule protector 16 modified molecule 17 biotin modified myoglobin

In the present specification, protein refers to a compound in which amino acids are peptide-bonded, and may be a natural protein or an artificially synthesized non-natural protein. In addition, the protein may include a natural modification. This protein may be, for example, an oligopeptide consisting of 10 or more amino acids, or a polypeptide having a higher order structure having a molecular weight of 5000 or more. In particular, in the present specification, a protein may refer to a protein that has a unique structure due to its post-translational folding, and that can exhibit some function / activity mainly in vivo.

The present invention is a method for obtaining a modified protein by binding a molecule having a desired chemical functional group to a protein by Pictet-Spengler reaction.
Hereinafter, the present invention will be described in more detail. In the following description, the part contributing to the Pictet-Spengler reaction at the end of a modified molecule to be introduced into a protein is referred to as a coupling partner. Moreover, the part introduced into the protein and specifically reacting with the coupling partner is referred to as a chemical handle.

According to the method for producing a modified protein in the present invention, a protein containing a chemical handle and a modified molecule containing a coupling partner are subjected to a Pictet-Spengler reaction under a weakly acidic to weakly basic condition, and the protein is converted into the modified molecule. It is a method to modify with.

<Picte Spengler reaction>
In the present invention, the Pictet-Spengler reaction refers to the following case.
When reacting a protein in which an ethylamine group having an aromatic ring or heteroaromatic ring at the β-position as a chemical handle is introduced with a modified molecule having an aldehyde group as a coupling partner at the end of the modifying unit.
When a protein having an aldehyde group introduced as a chemical handle is reacted with a modified molecule having an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position as a coupling partner at the end of the modification unit.
Alternatively, when a protein having an aldehyde group introduced as a chemical handle is reacted with a modified molecule composed of ethylamine having an aromatic or heteroaromatic ring at the β-position.
As a protein having an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position, a protein having a tryptophan residue at the N-terminus or C-terminus can also be used.

Here, the Pictet-Spengler reaction is a reaction in which a reaction between a chemical handle-containing protein and a modifying molecule having a coupling partner forms a carbon-carbon bond with ring-closing condensation after the reaction.
The Pictet-Spengler reaction uses a stable chemical handle and coupling partner, has a stable carbon-carbon bond generated by ring-closing condensation, has a high functional group specificity in the modification reaction, and reacts near room temperature. It is preferable at the point which progresses.
Here, the ring-closing condensation means that a heterocyclic skeleton is formed by the condensation of ethylamine having an aromatic ring or a heteroaromatic ring at the β-position with an aldehyde. Examples of the heterocyclic skeleton include a tetrahydrocarboline skeleton. When a reaction that forms a heterocyclic skeleton is used, a chemically stable carbon-carbon bond is formed as described above. Therefore, the modified product is difficult to be chemically decomposed. After the reaction, there is also an advantage that a function such as a fluorescent group can be added by further oxidizing the formed heterocyclic skeleton.

A typical Pictet-Spengler reaction is a chemical reaction in which a heterocyclic compound is formed as shown by the following general formula (7) in the presence of an acid catalyst such as HCl or TFA.

In general, the Pictet-Spengler reaction generates an iminium cation by dehydration of an amino group of an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position, represented by an indole-3-ethylamine skeleton, and the like, and an aldehyde group. This is the Mannich reaction type reaction in which the aromatic ring in the molecule causes Friedel-Crafts type nucleophilic addition. Therefore, depending on the protein or molecule to be used, it is necessary to advance the reaction by heating in an organic solvent under strongly acidic conditions. However, proteins are easily denatured under these conditions.
Therefore, when the Pictet-Spengler reaction is performed under mild conditions in which the protein is not denatured, the reactivity of indole-3-ethylamine and monoaldehyde groups is low, and the reactivity considered to be derived from steric hindrance on the protein. Due to the decrease, there is a problem that the modification of the protein hardly proceeds depending on the protein or the modifying molecule used.

The present inventors have found that in the Pictet-Spengler reaction, this reactivity is improved by optimizing the skeleton of an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position.
That is, it is preferable that an electron donating functional group is arranged on the aromatic ring or heteroaromatic ring of an ethylamine group having an aromatic ring or heteroaromatic ring at the β-position. Reactivity of Pictet-Spengler reaction under mild conditions that can suppress protein degradation or denaturation, such as pH 4-9, reaction temperature 0 ° C to 37 ° C Can be improved.

Examples of the electron-donating functional group include an alkyl group, an alkoxy group, an amino group, and a hydroxy group.
Examples of an ethylamine group having an aromatic ring or heteroaromatic ring at the β-position, and the above-mentioned electron-donating functional group arranged on the aromatic ring or heteroaromatic ring include, for example, 5 A pyrrole-3-ethylamine compound having an electron donating functional group at the 4-position or 6-position, and an electron-donating functional group at the 3-position or 5 And indole-3-ethylamine compound at the position.

Moreover, it is preferable that the carbon atom adjacent to the amine group of the ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position has an electron-withdrawing functional group. The electron withdrawing property of the iminium cation is improved, and nucleophilic addition is facilitated. Therefore, the reactivity of the Pictet-Spengler reaction can be improved.
Examples of the electron-withdrawing functional group include an imino group, a carbonyl group, and a carboxyl group.

Examples of such compounds having an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position include the following. For example, examples of the compound having a β-arylethylamine group include L-dopa (DOPA). Examples of the compound having an indole-3-ethylamine group include 6-hydroxytryptamine, tryptophan methyl ester, N-terminal tryptophan derivative and the like.

Typical skeletons of ethylamine groups having an aromatic ring or heteroaromatic ring at the β-position that are preferable for this method are listed in the following general formulas (8) to (10). The general formula (8) is a pyrrole-3-ethylamine compound, the general formula (9) is an indole-3-ethylamine compound, and the general formula (10) is a β-arylethylamine compound.

In general formula (8), R ¹ is preferably an electron-withdrawing functional group. R ⁴ is preferably an electron donating functional group. R ² and R ³ are not particularly limited, and examples thereof include an alkyl group, a hydroxy group, an alkoxy group, and an amino group. In the compound represented by the general formula (8), it is preferable that R ¹ to R ³ are bound to a modification unit or a protein.

In general formula (9), R ¹ is preferably an electron-withdrawing functional group. R ³ and R ⁵ are preferably electron donating functional groups. R ² , R ⁴ , and R ⁶ are not particularly limited, and examples thereof include an alkyl group, a hydroxy group, an alkoxy group, and an amino group. In the compound represented by the general formula (9), it is preferable that R ¹ , R ² , R ⁴ , and R ⁶ are bonded to the modification unit or the protein.

In general formula (10), R ¹ is preferably an electron-withdrawing functional group. R ⁴ and R ⁶ are preferably electron donating functional groups. R ² , R ³ , and R ⁵ are not particularly limited, and examples thereof include an alkyl group, a hydroxy group, an alkoxy group, and an amino group. In the compound represented by the general formula (10), it is preferable that R ¹ , R ² , R ³ and R ⁵ are bound to the modification unit or the protein.

In addition, the present inventors have found that the reactivity is improved by optimizing the aldehyde group in the Pict-Pengler reaction. That is, when the carbon atom adjacent to the aldehyde group has an electron-withdrawing group, nucleophilic addition is likely to proceed in the Pict-Pengler reaction. Examples of the electron-withdrawing functional group include an imino group, a carbonyl group, and a carboxyl group.

In the present invention, any aldehyde group can be used, but it is preferable to use an activated aldehyde. Specifically, for example, α-ketoaldehyde and the like can be mentioned.

In the present invention, the weakly acidic to weakly basic conditions for carrying out the Pictet-Spengler reaction are specifically pH 4.5 or more and pH 8.5 or less, preferably pH 5.0 or more and pH 6.5 or less. Moreover, phosphoric acid, acetic acid, etc. can be used as an acid catalyst.

As a solvent for carrying out the Pictet-Spengler reaction, in addition to an aqueous solvent, a solvent commonly used in organic reactions and complex synthesis, such as n-pentane, i-pentane, n-hexane, n-decane, benzene, toluene, acetone, Diethyl ether, diisopropyl ether, n-butyl methyl ether, t-butyl methyl ether, di-n-butyl ether, tetrahydrofuran, dioxane, N-methylpyrrolidone, N, N-dimethylformamide, dimethyl sulfoxide, triethylamine, hexamethylphosphoric acid A solvent such as triamide or a mixture thereof may be mentioned.

The reaction temperature is 4 ° C or higher and 80 ° C or lower, preferably 20 ° C or higher and 37 ° C or lower.

For example, an example of the modification reaction system in the present invention using the Pictet-Spengler reaction is as follows. A protein having a chemical handle of 1 μg / ml to 2000 μg / ml, preferably about 1000 μg / ml, and a molecule terminated with a coupling partner of 25 μM to 100 mM, preferably about 100 mM, 25 mM to 400 mM, preferably about 100 mM The pH is mixed in a buffer (sodium phosphate) at pH 4.5 to 8.5, preferably pH 5.5, the reaction temperature is 4 ° C. to 37 ° C., preferably 37 ° C., and the reaction time is 30 minutes to overnight. And incubate. This reaction yields a modified protein.

As described above, by using a combination of a highly reactive nucleophilic aromatic ring and an activated aldehyde, protein modification can be efficiently performed efficiently under weakly acidic to weakly basic conditions.
Specifically, an α-ketoaldehyde group, which is an active aldehyde with increased electrophilicity of an aldehyde, and a tryptamine methyl ester skeleton, which is an aromatic with increased nucleophilicity of indole-3-ethylamine, or indole- In order to enhance the nucleophilicity of 3-ethylamine, an electron-withdrawing functional group such as a carbonyl group is inserted at the α-position of the amino group. Accordingly, the reactivity of the Pictet-Spengler reaction is improved, and the protein modification reaction can proceed under mild conditions that can suppress degradation or denaturation of the protein. As a result, protein modification can be performed under weakly acidic to weakly basic conditions regardless of the three-dimensional structure of the protein and the modifying molecule used.

<Chemical handle-containing protein>
The protein is not particularly limited as long as it has a chemical handle, and a natural protein can be used, or a synthetic protein into which a chemical handle has been introduced by a conventionally known method can also be used.

The chemical handle is not particularly limited as long as it undergoes Picté-Spengler reaction with the coupling partner of the modifying molecule. When the coupling partner is an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position The chemical handle is an aldehyde group. When the coupling partner is an aldehyde group, the chemical handle is an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position. When using an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position as a chemical handle, a protein having an N-terminal tryptophan can also be used by a known method.

<Preparation of protein containing chemical handle>
Next, a method for introducing a chemical handle into a protein will be described.
The method for producing the chemical handle-containing protein is not particularly limited. For example, post-translational modification by oxidative deamination using pyridoxal-5-phosphate, a system for synthesizing a protein in an eubacteria or eukaryotic cell, Cell protein synthesis systems, post-translational modifications in vivo, and the like are all applicable. Alternatively, the N-terminus of the protein may be digested with any protease to obtain glycine, and then aldehyded by a known method.
In these chemical handle-containing protein synthesis methods, in order to bind molecules in a position-specific manner, the amino acid introduction position of an unnatural amino acid (amino acid having a chemical handle) is strictly controlled, or the position selectivity is high. Preferably, the chemical handle is made using an organic chemical reaction.

When an aldehyde group is introduced into a protein as a chemical handle, the aldehyde group can be introduced into an amino acid residue such as glycine or ε-N- (2-aminoacetyl) lysine by an oxidative deamination reaction.

The non-natural amino acid to be introduced into the protein is not particularly limited as long as it has the above chemical handle or can be used as a substrate for the Picter-Spengler reaction by functional group conversion. Examples of such a compound include p-aminoethylphenylalanine and δ-ketridine.

Since the chemical handle-containing protein can be easily produced at low cost, it is preferably produced by oxidative deamination using pyridoxal-5-phosphate. By treating the protein with the pyridoxal-5-phosphate, the N-terminus of the protein can be conveniently aldehyded by oxidative deamination.

In addition, a suppressor tRNA bound to an unnatural amino acid containing an ethylamine group or an aldehyde group having an aromatic ring or a heteroaromatic ring at the β-position; and a nonsense mutation was performed so as to have a codon corresponding to the codon of the suppressor tRNA It is preferable to express a chemical handle-containing protein by a synthesis system using a gene and; Unnatural amino acids can be introduced in a site-specific manner in the protein.

As the mutant aminoacyl tRNA synthetase, an aminoacyl tRNA synthetase having substrate specificity for a non-natural amino acid containing a functional group capable of Pictet-Spengler reaction is used. Alternatively, a mutant aminoacyl-tRNA synthetase having enhanced substrate specificity for a non-natural amino acid containing a functional group capable of Pictet-Spengler reaction compared to the original substrate specificity for an amino acid can also be used. Such a mutant aminoacyl tRNA synthetase can be appropriately selected according to the unnatural amino acid to be used, and examples thereof include tyrosyl-tRNA synthetase, tryptophanyl-tRNA synthetase, and pyrrolidyl-tRNA synthetase.

A suppressor tRNA is a tRNA that is aminoacylated with an aminoacyl-tRNA synthetase or a mutant thereof with an unnatural amino acid containing a functional group capable of Pictet-Spengler reaction, and whose anticodon corresponds to a stop codon.
The suppressor tRNA can be prepared by preparing a template DNA having a sequence to serve as a template for transcription by a known method and performing the transcription reaction.

The method of providing a nonsense mutation at a desired position of a gene is not particularly limited, and can be performed by the methods described in Non-Patent Documents 1 to 11, for example.

By using these aminoacyl tRNA synthetases, suppressor tRNAs, and genes that have undergone a nonsense mutation at a desired position, for example, unnatural amino acids such as p-aminoethylphenylalanine and δ-ketridine can be introduced into proteins (for example, (See Non-Patent Documents 1 to 11).

In addition, a protein having an aldehyde group can be produced by digesting a natural protein or a synthesized protein with a protease in which the N-terminus is a glycine residue. For example, an expression vector incorporating a GST (glutathione-S-transferase) tag and a gene modified so that the downstream of the recognition site of Factor Xa is a glycine residue is transfected into E. coli or cultured cells. And recover proteins expressed in cultured cells. And the protein which has a glycine residue in N terminal can be obtained by digesting this collect | recovered protein with Factor Xa.

<Coupling partner-containing modified molecule>
The modified molecule refers to a molecule in which a coupling partner is bound to the end of the modification unit. When the coupling partner is an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position, the modifying unit is, for example, R ¹ , R ² of the β-arylethylamine group represented by the general formula (4) described above, R ³ and R ⁵ are preferably bonded. When the coupling partner is an aldehyde group, the modification unit is preferably bonded to a carbon atom of the aldehyde group.

The modification unit is not particularly limited, and examples thereof include amines (first, second, third, and fourth), alcohols (first, second, and third), carbonyl groups, carboxyl groups, thiol groups, and sulfoxide groups. , A functional group containing a functional group such as a sulfone group, a sulfonic acid, an aryl group, an allyl group, a sulfur-containing heterocycle, a nitrogen-containing heterocycle, or a condensate thereof. In addition, known coenzymes, sugar chains, fatty chains, DNA, RNA, nucleosides, nucleotides, proteins, peptides, lipids, carbohydrates and derivatives thereof, or non-natural molecules can be used. Furthermore, as the modification unit, a fluorescent molecule, a well-known molecule (liquid crystal molecule) showing liquid crystal, a substrate, fullerene, or the like can be used.
It is also possible to use a molecule that can bind to a glass substrate or a resin substrate as a modification unit, and to bind a protein to these substrates via this modification unit and a coupling partner. In this case, the protein can be bound to the substrate by binding the modifying molecule comprising the modifying unit and the coupling partner to the substrate first, and then binding the protein to the modifying molecule by Pictet-Spengler reaction.

<Preparation of modified molecules containing coupling partners>
For example, an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position can be added as a coupling partner to the terminal of a modification unit to be introduced into a protein. Examples of a method for adding an ethylamine group having an aromatic ring or heteroaromatic ring at the β-position as a coupling partner to the modification unit include tryptophan represented by the following general formula (11) or L-DOPA represented by the general formula (12). Are covalently bonded to the modifying unit by a condensation reaction. These tryptophan, L-DOPA, etc. can use a commercial item.

In the general formula (11), R is a modification unit.

In the general formula (12), R is a modification unit.

Alternatively, an aldehyde group can be added as a coupling partner to the end of the modification unit. As a method for introducing an aldehyde group as a coupling partner, for example, 2-aminoacetamide is converted to an aldehyde group by oxidative deamination reaction or a hydroxy group is converted by oxidation reaction.

Thus, a modified molecule having a coupling partner at the terminal is obtained. A modification unit having a coupling partner in advance can be used as a modification molecule, or the coupling partner itself can be used as a modification molecule.

An example of the aspect of the present invention is as shown in FIG. In FIG. 1, horse heart myoglobin (hereinafter sometimes referred to as myoglobin) is used as a protein. In FIG. 1, the aldehyde group arranged at the N-terminus of myoglobin as a chemical handle and a tryptamine derivative (for example, tryptophan methyl ester) having indole-3-ethylamine as a coupling partner were subjected to Pictet-Spengler reaction. The case is illustrated. However, proteins, chemical handles, and coupling partners are not limited to this combination.

FIG. 1 shows that myoglobin 1 having an aldehyde group at the N-terminus (formylated myoglobin 1) and tryptamine 2 having indole-3-ethylamine are converted to tryptamine-modified myoglobin 3 by the Pictet-Spengler reaction. Thereafter, oxidation may result in β-carboline-modified myoglobin 4. Thus, by oxidizing the heterocyclic skeleton, a function as a fluorescent group can be added.

<Purification of modified protein>
Confirmation of the product (modified protein) can be detected by SDS-PAGE, mass spectrometry or the like.
For the isolation and purification of the modified protein, for example, solvent extraction, fractional precipitation with an organic solvent, salting out, dialysis, centrifugation, ultrafiltration, ion exchange chromatography, gel filtration chromatography. Separation operations such as chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, crystallization, and electrophoresis can be performed alone or in combination.
The fact that the modified protein is not cleaved / degraded can be confirmed by SDS-PAGE separation and subsequent detection with an antibody specific to the modified protein.
Further, the activity of the modified protein can be confirmed by a well-known method for the modified protein (for example, if the activity is retained, it is understood that the modified protein has the correct three-dimensional structure).
Moreover, it can confirm that the activity of the introduce | transduced molecule | numerator is hold | maintained by investigating the activity of this molecule | numerator. For example, when biotin is bound, the binding ability to avidin can be confirmed, and when a fluorescent substance is bound, it can be confirmed by fluorescence observation.

Thus, in the present invention, various reactions are possible by combination with known methods.

Examples of the modified protein obtained by the method for producing a modified protein of the present invention include those represented by the following general formula (13).

In the general formula (13), R ¹ is a protein, R ⁵ is a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and at least one of R ² , R ³ , and R ⁴ One is the modification unit, the rest are R ² is an imino group, a carbonyl group, and a carboxyl group, and R ³ and R ⁴ are each independently a hydrogen atom, an alkyl group, a hydroxy group, an alkoxy group, or an amino group. is there. More specifically, for example, R ¹ is a protein, R ² is a modification unit, and R ³ , R ⁴ , and R ⁵ are hydrogen atoms, and are modified proteins represented by the following general formula (14). The modification unit uses biotin with amide as a linker. The resulting modified protein is modified with biotin.

Moreover, the following can be mentioned as a modified protein.

In the general formula (15), R ¹ is a protein, R ⁵ is a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and at least one of R ² , R ³ , and R ⁴ One is the modification unit, the rest are R ² is an imino group, a carbonyl group, and a carboxyl group, and R ³ and R ⁴ are each independently a hydrogen atom, an alkyl group, a hydroxy group, an alkoxy group, or an amino group. is there. More specifically, for example, R ¹ is a protein and R ² to R ⁵ are hydrogen atoms, and is a modified protein represented by the following general formula (16). The modified molecule consists of ethylamine having an aromatic ring at the β-position, and the resulting protein is modified with 6-azaindole (fluorescent group).

Moreover, the following can be mentioned as a modified protein.

In the general formula (17), R ¹ is a protein, R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁵ and R ⁷ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁵ , and R ⁷ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group. More specifically, for example, R ¹ is a protein, R ² is a modification unit, and R ³ to R ⁷ are hydrogen atoms, and are modified proteins represented by the following general formula (18). The modification unit uses biotin with amide as a linker. The resulting modified protein is modified with biotin.

Moreover, the following can be mentioned as a modified protein.

In the general formula (19), R ¹ is a protein, R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁵ and R ⁷ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁵ , and R ⁷ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group. More specifically, for example, R ¹ is a protein, and R ² to R ⁷ are hydrogen atoms, and are modified proteins represented by the following general formula (20). The modifying molecule is composed of ethylamine having a heteroaromatic ring at the β-position, and the resulting protein is modified with norharman (fluorescent group).

Moreover, the following can be mentioned as a modified protein.

In the general formula (21), R ¹ is a protein, R ⁵ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁴ and R ⁶ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁴ , and R ⁶ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group. More specifically, for example, R ¹ is a protein, R ² is a modification unit, R ⁵ is a hydroxy group, and R ³ , R ⁴ , R ⁶ , and R ⁷ are hydrogen atoms. (22) is a modified protein. The modification unit uses biotin with amide as a linker. The resulting modified protein is modified with biotin.

Moreover, the following can be mentioned as a modified protein.

In the general formula (23), R ¹ is a protein, R ⁵ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁴ and R ⁶ are at least one of the modification units, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁴ , and R ⁶ are each independently a hydrogen atom. An alkyl group, a hydroxy group, an alkoxy group, and an amino group. More specifically, for example, R ¹ is a protein, and R ² to R ⁷ are hydrogen atoms, and are modified proteins represented by the following general formula (24). The modified molecule is composed of ethylamine having an aromatic ring at the β-position, and the resulting modified protein is modified with isoquinoline (fluorescence quenching group).

In addition to the modified proteins exemplified above, various types as shown below can be used as modified molecules, and application examples as shown below can be considered.
(1) Labeled protein For example, a modified protein obtained by binding a fluorescent molecule such as fluorescein, Alexa Fluor, or Bodypi (BODIPY) via a coupling partner is preferably used for analyzing the behavior of the protein. . In the past, proteins with such fluorescent molecules as labels, such as GFP, have been known. However, the method of the present invention can introduce fluorescent molecules in a position-specific manner for proteins. Therefore, it can be selectively introduced at a location that does not affect the activity of the protein. In addition, steric hindrance that inhibits protein function hardly occurs. Therefore, more accurate analysis of protein behavior is possible. A protein into which a molecule such as biotin is introduced can also be preferably used for analyzing the behavior of the protein.
FIG. 2 is a diagram showing an example of a protein into which a fluorescent molecule is introduced. Fluorescein-modified myoglobin 6 into which fluorescein has been introduced is obtained by carrying out a Pictet-Spengler reaction between formylated myoglobin 1 and modified molecule 5 containing fluorescein.

(2) Detection of interaction target of unknown function protein By the method of the present invention, a fluorescent group is introduced into the unknown function protein to obtain a labeled protein. This is made to exist in a cell with a cDNA library, and FRET (fluorescence resonance energy transfer) is measured. Here, it is suggested that the protein expressed from the library or its effector protein may interact with the labeled protein in the cells in which the change in the fluorescence property is detected. As the fluorescent group, fluorescein, alexafluoro, body pie, rhodamine, harmine and the like can be used. FIG. 3 is a diagram showing an example of a protein into which a fluorescent group has been introduced. By performing the Pictet-Spengler reaction between the formylated myoglobin 1 and the modified molecule 7 containing Alexafluoro488, Alexafluoro488-modified myoglobin 8 into which the modified molecule 7 containing Alexafluoro488 has been introduced is obtained.

(3) Binding liquid crystal molecules Proteins introduced with liquid crystal molecules are aligned in a high magnetic field by the action of the liquid crystal molecules. By utilizing this, structural analysis by NMR becomes easy, and high throughput of structural analysis can be achieved. FIG. 4 is a diagram showing an example of a protein into which liquid crystal molecules are introduced. MBBA-modified myoglobin 10 into which MBBA 9 has been introduced is obtained by performing a Pictet-Spengler reaction between formylated myoglobin 1 and a modified molecule 9 containing methoxybenzylidene p-butyl aniline (MBBA) as a liquid crystal molecule. Is obtained.

(4) Introduction of heavy atoms into protein Crystallization after introducing heavy atoms into protein enables phase determination. Structural analysis is facilitated because the labor for preparing heavy atom isomorphous substituents can be saved. FIG. 5 is a diagram showing an example of a protein having a bromine atom introduced as a heavy atom. By performing the Pictet-Spengler reaction between the formylated myoglobin 1 and the modified molecule 11 containing a bromine atom, a heavy element modified myoglobin 12 in which a bromine atom is introduced as a heavy element is obtained.

(5) Immobilization of protein on the chip When the protein is immobilized on the chip, the in-vivo system can be reconfigured on the chip. According to the method of the present invention, the site of protein fixation to a chip can be performed site-specifically, and protein orientation can be controlled. Such a protein chip is useful for searching for a ligand.

(6) Drug delivery system A drug that binds to a substance that is localized at a pathogenic site and a drug are bound by the method of the present invention, and the drug can be administered to localize the drug at a desired pathogenic site. . Therefore, the medication effect can be improved.

(7) Enzyme stabilization by modification with polyethylene glycol (PEG) By PEGylating a protein, it becomes difficult to be degraded by protease. In addition, the higher-order structure of the protein is stabilized, and the improvement of the enzyme function is expected. Therefore, by the method of the present invention, by introducing site-specific PEG, the protein can be more stabilized without impairing the activity of the protein.

(8) Introduction into photoaffinity labeled probe The photoaffinity labeled probe is a technique widely used for identification of a target protein which is a physiologically active substance. By introducing α-ketoaldehyde into the probe, photolabeling, and then using the present invention, it becomes possible to isolate or detect only the labeled protein.

<Protein modification kit>
By using a protein modification kit to which the method for producing a modified protein of the present invention is applied, a modified protein can be obtained more easily. For example, a protein modification kit is roughly composed of pyridoxal-5-phosphate and a β-arylethylamine group-containing modified molecule.
A modified protein is obtained by mixing pyridoxal-5-phosphate and a β-arylethylamine group-containing modified molecule with the protein to be modified and reacting at 24 ° C. or higher and 37 ° C. or lower, for example, overnight.

The following examples are for explaining the present invention more specifically, and the scope of the present invention is not limited by these examples.
When handling proteins, all operations were performed under ice cooling unless otherwise noted. The pH in the buffer or the like was adjusted based on the pH meter and the conditions described in the literature. As the water, ultrapure water (made by Milli-Q) was used.

<Example 1>
(1) Preparation of α-ketoaldehyde-ized myoglobin According to Non-Patent Document 24, myoglobin was dissolved in a phosphate buffer (25 mM, pH 6.5) to give a 100 μM solution. An equal amount of pyridoxal-5-phosphate / phosphate buffer solution was added to this solution and incubated at 37 ° C. for 24 hours. The myoglobin solution in which the N-terminal was aldehyded by the reaction was purified by buffer exchange using ultrafiltration by centrifugation at 12,000 rpm.

(2) Synthesis of Biotinylated Detection Marker Unit In order to introduce biotin 13 into a protein, modified molecule 16 bound to biotin 13 was synthesized using tryptophan 14 as a coupling partner by the following method. This process is shown in FIG.
(A) Synthesis of modified molecular protector 15 by dehydration condensation of tryptophan 14 and biotin 13 31.2 mg synthesized according to the literature (Wilbur et al., Bioconj. Chem. Vol. 7, 1996, p. 689-702) Biotin 13 was dissolved in 1 ml of dichloromethane and cooled to 0 ° C. 1 ml of TFA was dissolved in this solution and stirred at 0 ° C. for 3 hours. Thereafter, the reaction solution was distilled off under reduced pressure using an evaporator. The residue was dissolved in 2.0 ml DMF, 55 mg (Boc) 2 Tryptophan 14, 5.5 mg HOBt, 77 mg EDC and 200 μl DIEA were added to the solution and stirred at room temperature for 18 hours. The reaction solution was azeotroped with toluene, and the solvent was distilled off under reduced pressure. The two layers were partitioned between dichloromethane and water, and the organic layer was dried over magnesium sulfate and then distilled off under reduced pressure. By purifying by silica gel chromatography (ethyl acetate → ethyl acetate: methanol = 10: 1), 45.0 mg of the modified molecular protector 15 into which the target Boc-protected tryptophan represented by the following general formula (25) was introduced, The yield was 93%.

(B) Synthesis of Modified Molecule 16 by Deprotection of Boc Group 10 mg of the modified molecular protector 15 obtained above was dissolved in 1 ml of dichloromethane and cooled to 0 ° C. 1 ml of TFA was dissolved in this solution and stirred at 0 ° C. for 3 hours. Thereafter, the reaction solution was distilled off under reduced pressure using an evaporator. The residue was dissolved in dichloromethane, and TFA was distilled off by repeating air drying twice. Thereafter, purification by reverse layer chromatography using ODS yielded 6.6 mg of the target modified molecule 16 represented by the following general formula (26) in a yield of 90%.

(3) Modification of protein Each composition of the reaction system is as follows. 2.0 mg / ml protein (α-ketoaldehyde horse horse myoglobin 1), 10 mM modified molecule 16 prepared above and 100OmM buffer (sodium phosphate, pH 6.5). These reagents were mixed and then incubated at 37 degrees for 18 hours to obtain the product. As shown in FIG. 7, the obtained product is considered to be biotin-modified myoglobin 17 modified with biotin.

<Comparative example>
(1) Competitive inhibition using methoxyamine 160 mM methoxyamine was added to the above-described reaction solution, and a modification reaction was performed under the condition of competitive inhibition.

<Detection of product>
The products obtained in Example 1 and the comparative example were immediately desalted by gel filtration, separated by SDS-PAGE, and detected by cyprotangerin staining and Western blotting.
The result is shown in FIG.
In FIG. 8, lane 1 is the protein migrated in Comparative Example 1, lane 2 is the protein migrated in Example 1, lane 3 is the one that migrates only myoglobin, lane 4 is biotinylated Only the detection marker unit 2 is electrophoresed. The image shown in the upper part of FIG. 8 is the result of cyprotangerin staining, and the image shown in the lower part is the result of Western blotting using an anti-biotin antibody.
From the results in the upper part of FIG. 8, it was found that the product of Example 1 produced a single 17 kDa band on the SDS polyacrylamide gel, and thus was not subjected to oxidative degradation. Furthermore, from the results shown in the upper and lower parts of FIG. 8, it was concluded that this band is a modified protein 3 in which biotin is coupled to myoglobin since it gives a positive signal even if it is detected by a well-known detection method for biotin. .
From FIG. 8, in the product of the comparative example, the band detected by cyprotangerin staining did not give a positive signal even when detected by the detection method for biotin. Therefore, it was considered that the modification was inhibited.

<Example 2>
(1) Preparation of α-ketoaldehyde-ized myoglobin (1) According to [Non-patent Document 24], horse heart myoglobin was dissolved in 25 mM phosphate buffer (pH 6.5) to give a 100 μM solution. Then, an equal amount of pyridoxal-5-phosphate / phosphate buffer solution was added to this solution and incubated at 37 ° C. for 24 hours. The myoglobin solution in which the N terminus was aldehyded by the reaction was purified by buffer exchange using ultrafiltration by centrifugation at 12,000 rpm.

(2) Protein modification reaction The following composition was used as a reaction system. 2.0 mg / ml protein (α-ketoaldehyde myoglobin), 100 mM tryptamine, or tryptophan methyl ester, 100 mM buffer (sodium phosphate, pH 6.5). These reagents were mixed and then incubated at 37 ° C. for 18 hours to obtain the product.

The product obtained above was subjected to a desalting operation and then measured using mass spectrometry (MALDI-TOF MS, ESI-TOF MS). The result is shown in FIG.
From FIG. 9, since full-length ion peaks corresponding to tryptamine-modified myoglobin and tryptophan methyl ester-modified myoglobin were detected, the above products were tryptamine-modified myoglobin shown in FIG. 10A and tryptophan methyl ester-modified myoglobin shown in FIG. 10B. It was confirmed that the modification of myoglobin was performed. Further, from the relative intensity shown in FIG. 9, it is estimated that about 1/3 of myoglobin was labeled.

In addition, site-specific modification was confirmed by MS / MS analysis. The result is shown in FIG.
FIG. 11 confirmed that the N-terminal glycine was chemoselectively modified. Precursor ions are presumed to be oxidized during mass spectrometry to form a harmine skeleton.

Furthermore, the presence or absence of denaturation was confirmed by UV measurement and CD measurement of the modified myoglobin. The results are shown in FIGS. FIG. 12 shows the UV measurement results, and FIG. 13 shows the CD measurement results.
From FIG. 12, since the absorption peak intensity around 280 nm derived from the indole skeleton was increased, it was confirmed that the indole skeleton was imparted by the modification. Further, no change was observed in the absorption peak derived from hemin near 400 nm even after modification.
From FIG. 13, no difference was observed in the absorption peak derived from α-helix between wild-type myoglobin and modified myoglobin.
From the above, it was suggested that the modified myoglobin was not denatured.

The present invention can be applied to adding various modifying molecules to proteins.

Claims (13)

1. A protein containing an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position, and a modified molecule having an aldehyde group at the end of the modifying unit,
   A protein containing an aldehyde group and a modified molecule having an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position at the end of the modifying unit, or
   A protein containing an aldehyde group and a modified molecule composed of ethylamine having an aromatic ring or a heteroaromatic ring at the β-position,
   A method for producing a modified protein, characterized in that a Pictet-Spengler reaction is performed under mildly acidic to weakly basic conditions, and the protein is modified with the modified molecule closed.
2. The method for producing a modified protein according to claim 1, wherein the aromatic ring or heteroaromatic ring has an electron-donating functional group.
3. The method for producing a modified protein according to claim 2, wherein the electron-donating functional group is one or more selected from the group consisting of an alkyl group, an alkoxy group, an amino group, and a hydroxy group.
4. The method for producing a modified protein according to claim 1, wherein the carbon atom adjacent to the amine group of the ethylamine group has an electron-withdrawing functional group.
5. The method for producing a modified protein according to claim 1, wherein a carbon atom adjacent to the aldehyde group has an electron-withdrawing functional group.
6. 6. The method for producing a modified protein according to claim 4, wherein the electron-withdrawing functional group is at least one selected from the group consisting of an imino group, a carbonyl group and a carboxyl group.
7. The method for producing a modified protein according to claim 1, wherein the ethylamine group having an aromatic ring or a heteroaromatic ring forms a tryptamine skeleton or a dopamine skeleton, and the aldehyde group forms an α-ketoaldehyde.
8. The method for producing a modified protein according to claim 1, wherein the aldehyde group-containing protein can be introduced with an aldehyde group by oxidative deamination reaction in the presence of pyridoxal-5-phosphate.
9. The protein comprises a suppressor tRNA bonded to an amino acid containing an ethylamine group or an aldehyde group having an aromatic ring or heteroaromatic ring at the β-position; and a gene that has been subjected to nonsense mutation so as to have a codon corresponding to the codon of the suppressor tRNA The method for producing a modified protein according to claim 1, wherein the protein is synthesized by a synthesis system using
10. The method for producing a modified protein according to claim 1, wherein the protein containing an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position is a protein having a tryptophan residue at the N-terminus.
11. A modified protein obtained by the method for producing a modified protein according to claim 1.
12. The modified protein according to claim 11, which is represented by any one of the following general formulas (1) to (6).
    

    

    (In the above general formula, R ¹ is a protein, R ⁵ is a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and at least one of R ² , R ³ , and R ^4. Is the above-mentioned modification unit, and the remaining R ² is an imino group, a carbonyl group or a carboxyl group, and R ³ and R ⁴ are each independently a hydrogen atom, an alkyl group, a hydroxy group, an alkoxy group or an amino group. )
    

    

    (In the above general formula, R ¹ is a protein, R ⁵ is a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and at least one of R ² , R ³ , and R ^4. Is the above-mentioned modification unit, and the remaining R ² is an imino group, a carbonyl group or a carboxyl group, and R ³ and R ⁴ are each independently a hydrogen atom, an alkyl group, a hydroxy group, an alkoxy group or an amino group. )
    

    

    (In the above general formula, R ¹ is a protein, R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁵ , At least one of R ⁷ is the modification unit, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁵ , and R ⁷ are each independently a hydrogen atom, alkyl Group, hydroxy group, alkoxy group and amino group.)
    

    

    (In the above general formula, R ¹ is a protein, R ⁴ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁵ , At least one of R ⁷ is the modification unit, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁵ , and R ⁷ are each independently a hydrogen atom, alkyl Group, hydroxy group, alkoxy group and amino group.)
    

    

    (In the above general formula, R ¹ is a protein, R ⁵ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁴ , At least one of R ⁶ is the modification unit, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁴ , and R ⁶ are each independently a hydrogen atom, alkyl Group, hydroxy group, alkoxy group and amino group.)
    

    

    (In the above general formula, R ¹ is a protein, R ⁵ and R ⁶ are each independently a hydrogen atom, an alkyl group, an alkoxy group, an amino group, or a hydroxy group, and R ² , R ³ , R ⁴ , At least one of R ⁶ is the modification unit, the remaining R ² is an imino group, a carbonyl group, or a carboxyl group, and R ³ , R ⁴ , and R ⁶ are each independently a hydrogen atom, alkyl Group, hydroxy group, alkoxy group and amino group.)
13. A protein modification kit for producing a modified protein, characterized by having pyridoxal-5-phosphate and a modified molecule containing an ethylamine group having an aromatic ring or a heteroaromatic ring at the β-position kit.

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